Inhibition of mTORC1 by astrin and stress granules prevents apoptosis in cancer cells.

Mammalian target of rapamycin complex 1 (mTORC1) controls growth and survival in response to metabolic cues. Oxidative stress affects mTORC1 via inhibitory and stimulatory inputs. Whereas downregulation of TSC1-TSC2 activates mTORC1 upon oxidative stress, the molecular mechanism of mTORC1 inhibition remains unknown. Here, we identify astrin as an essential negative mTORC1 regulator in the cellular stress response. Upon stress, astrin inhibits mTORC1 association and recruits the mTORC1 component raptor to stress granules (SGs), thereby preventing mTORC1-hyperactivation-induced apoptosis. In turn, balanced mTORC1 activity enables expression of stress factors. By identifying astrin as a direct molecular link between mTORC1, SG assembly, and the stress response, we establish a unifying model of mTORC1 inhibition and activation upon stress. Importantly, we show that in cancer cells, apoptosis suppression during stress depends on astrin. Being frequently upregulated in tumors, astrin is a potential clinically relevant target to sensitize tumors to apoptosis.

Adaptive digital tissue deconvolution.

MOTIVATION: The inference of cellular compositions from bulk and spatial transcriptomics data increasingly complements data analyses. Multiple computational approaches were suggested and recently, machine learning techniques were developed to systematically improve estimates. Such approaches allow to infer additional, less abundant cell types. However, they rely on training data which do not capture the full biological diversity encountered in transcriptomics analyses; data can contain cellular contributions not seen in the training data and as such, analyses can be biased or blurred. Thus, computational approaches have to deal with unknown, hidden contributions. Moreover, most methods are based on cellular archetypes which serve as a reference; e.g. a generic T-cell profile is used to infer the proportion of T-cells. It is well known that cells adapt their molecular phenotype to the environment and that pre-specified cell archetypes can distort the inference of cellular compositions. RESULTS: We propose Adaptive Digital Tissue Deconvolution (ADTD) to estimate cellular proportions of pre-selected cell types together with possibly unknown and hidden background contributions. Moreover, ADTD adapts prototypic reference profiles to the molecular environment of the cells, which further resolves cell-type specific gene regulation from bulk transcriptomics data. We verify this in simulation studies and demonstrate that ADTD improves existing approaches in estimating cellular compositions. In an application to bulk transcriptomics data from breast cancer patients, we demonstrate that ADTD provides insights into cell-type specific molecular differences between breast cancer subtypes. AVAILABILITY AND IMPLEMENTATION: A python implementation of ADTD and a tutorial are available at Gitlab and zenodo (doi:10.5281/zenodo.7548362).

The splicing landscape is globally reprogrammed during male meiosis.

Meiosis requires conserved transcriptional changes, but it is not known whether there is a corresponding set of RNA splicing switches. Here, we used RNAseq of mouse testis to identify changes associated with the progression from mitotic spermatogonia to meiotic spermatocytes. We identified ∼150 splicing switches, most of which affect conserved protein-coding exons. The expression of many key splicing regulators changed in the course of meiosis, including downregulation of polypyrimidine tract binding protein (PTBP1) and heterogeneous nuclear RNP A1, and upregulation of nPTB, Tra2ß, muscleblind, CELF proteins, Sam68 and T-STAR. The sequences near the regulated exons were significantly enriched in target sites for PTB, Tra2ß and STAR proteins. Reporter minigene experiments investigating representative exons in transfected cells showed that PTB binding sites were critical for splicing of a cassette exon in the Ralgps2 mRNA and a shift in alternative 5' splice site usage in the Bptf mRNA. We speculate that nPTB might functionally replace PTBP1 during meiosis for some target exons, with changes in the expression of other splicing factors helping to establish meiotic splicing patterns. Our data suggest that there are substantial changes in the determinants and patterns of alternative splicing in the mitotic-to-meiotic transition of the germ cell cycle.

PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects.

The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.

Molecular design of a splicing switch responsive to the RNA binding protein Tra2ß.

Tra2ß regulates a number of splicing switches including activation of the human testis-specific exon HIPK3-T in the Homeodomain Interacting Protein Kinase 3 gene. By testing HIPK3-T exons of different intrinsic strengths, we found Tra2ß most efficiently activated splicing inclusion of intrinsically weak exons, although these were spliced at a lower overall level. Both the RRM and N-terminal RS-rich region of Tra2ß were required for splicing activation. Bioinformatic searches for splicing enhancers and repressors mapped four physically distinct exonic splicing enhancers (ESEs) within HIPK3-T, each containing the known Tra2ß AGAA-rich binding site. Surprisingly disruption of each single ESE prevented Tra2ß-mediated activation, although single mutated exons could still bind Tra2ß protein by gel shifts and functional splicing analyses. Titration experiments indicate an additive model of HIPK3-T splicing activation, requiring availability of an array of four distinct ESEs to enable splicing activation. To enable this efficient Tra2ß-mediated splicing switch to operate, a closely adjacent downstream and potentially competitive stronger 5'-splice site is actively repressed. Our data indicate that a novel arrangement of multiple mono-specific AGAA-rich ESEs coupled to a weak 5'-splice site functions as a responsive gauge. This gauge monitors changes in the specific nuclear concentration of the RNA binding protein Tra2ß, and co-ordinately regulates HIPK3-T exon splicing inclusion.

FAIR+E pathogen data for surveillance and research: lessons from COVID-19.

The COVID-19 pandemic has exemplified the importance of interoperable and equitable data sharing for global surveillance and to support research. While many challenges could be overcome, at least in some countries, many hurdles within the organizational, scientific, technical and cultural realms still remain to be tackled to be prepared for future threats. We propose to (i) continue supporting global efforts that have proven to be efficient and trustworthy toward addressing challenges in pathogen molecular data sharing; (ii) establish a distributed network of Pathogen Data Platforms to (a) ensure high quality data, metadata standardization and data analysis, (b) perform data brokering on behalf of data providers both for research and surveillance, (c) foster capacity building and continuous improvements, also for pandemic preparedness; (iii) establish an International One Health Pathogens Portal, connecting pathogen data isolated from various sources (human, animal, food, environment), in a truly One Health approach and following FAIR principles. To address these challenging endeavors, we have started an ELIXIR Focus Group where we invite all interested experts to join in a concerted, expert-driven effort toward sustaining and ensuring high-quality data for global surveillance and research.

An apparent pseudo-exon acts both as an alternative exon that leads to nonsense-mediated decay and as a zero-length exon.

Pseudo-exons are intronic sequences that are flanked by apparent consensus splice sites but that are not observed in spliced mRNAs. Pseudo-exons are often difficult to activate by mutation and have typically been viewed as a conceptual challenge to our understanding of how the spliceosome discriminates between authentic and cryptic splice sites. We have analyzed an apparent pseudo-exon located downstream of mutually exclusive exons 2 and 3 of the rat alpha-tropomyosin (TM) gene. The TM pseudo-exon is conserved among mammals and has a conserved profile of predicted splicing enhancers and silencers that is more typical of a genuine exon than a pseudo-exon. Splicing of the pseudo-exon is fully activated for splicing to exon 3 by a number of simple mutations. Splicing of the pseudo-exon to exon 3 is predicted to lead to nonsense-mediated decay (NMD). In contrast, when "prespliced" to exon 2 it follows a "zero length exon" splicing pathway in which a newly generated 5' splice site at the junction with exon 2 is spliced to exon 4. We propose that a subset of apparent pseudo-exons, as exemplified here, are actually authentic alternative exons whose inclusion leads to NMD.

An ancient germ cell-specific RNA-binding protein protects the germline from cryptic splice site poisoning.

Male germ cells of all placental mammals express an ancient nuclear RNA binding protein of unknown function called RBMXL2. Here we find that deletion of the retrogene encoding RBMXL2 blocks spermatogenesis. Transcriptome analyses of age-matched deletion mice show that RBMXL2 controls splicing patterns during meiosis. In particular, RBMXL2 represses the selection of aberrant splice sites and the insertion of cryptic and premature terminal exons. Our data suggest a Rbmxl2 retrogene has been conserved across mammals as part of a splicing control mechanism that is fundamentally important to germ cell biology. We propose that this mechanism is essential to meiosis because it buffers the high ambient concentrations of splicing activators, thereby preventing poisoning of key transcripts and disruption to gene expression by aberrant splice site selection.

Nonsense-mediated messenger RNA decay of survival motor neuron 1 causes spinal muscular atrophy.

Autosomal recessive proximal spinal muscular atrophy (SMA) is a neurodegenerative disorder resulting from functional loss of survival motor neuron 1 (SMN1). Homozygous absence of SMN1 due to deletion or gene conversion accounts for about 96% of SMA cases. In the remaining 4%, subtle SMN1 mutations are commonly identified. Here, we describe two novel intragenic SMN1 mutations in three type I SMA individuals: a point mutation in exon 3 (c.469C > T) and a substitution in intron 4 (c.628-140A > G). In-vivo splicing assays demonstrated that the intronic substitution creates a novel splice donor site, culminating in aberrant splicing and insertion of 65 bp from intron 4 between exons 4 and 5 in SMN1 transcripts (c.627_628ins65). Both mutations render SMN1 transcripts susceptible to nonsense-mediated mRNA decay (NMD), resulting in mRNA degradation, insufficient SMN protein levels and development of an SMA phenotype. Treatment of patient cell lines with the translation inhibitors puromycin and emetine markedly increased the levels of mutant SMN1 transcripts. A similar effect was observed after siRNA-mediated knockdown of UPF1, a factor essential for NMD. This study provides first evidence that NMD of SMN1 transcripts is responsible for the molecular basis of disease in a subset of SMA patients.

Disrupted alternative splicing for genes implicated in splicing and ciliogenesis causes PRPF31 retinitis pigmentosa.

Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause non-syndromic retinal disease. Here, we generate transcriptome profiles from RP11 (PRPF31-mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as Prpf31+/- mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31+/- mouse retinae and RPE. Mis-splicing of genes implicated in ciliogenesis and cellular adhesion was associated with severe RPE defects that include disrupted apical - basal polarity, reduced trans-epithelial resistance and phagocytic capacity, and decreased cilia length and incidence. Disrupted cilia morphology also occurred in patient-derived photoreceptors, associated with progressive degeneration and cellular stress. In situ gene editing of a pathogenic mutation rescued protein expression and key cellular phenotypes in RPE and photoreceptors, providing proof of concept for future therapeutic strategies.

Cellular senescence drives age-dependent hepatic steatosis.

The incidence of non-alcoholic fatty liver disease (NAFLD) increases with age. Cellular senescence refers to a state of irreversible cell-cycle arrest combined with the secretion of proinflammatory cytokines and mitochondrial dysfunction. Senescent cells contribute to age-related tissue degeneration. Here we show that the accumulation of senescent cells promotes hepatic fat accumulation and steatosis. We report a close correlation between hepatic fat accumulation and markers of hepatocyte senescence. The elimination of senescent cells by suicide gene-meditated ablation of p16Ink4a-expressing senescent cells in INK-ATTAC mice or by treatment with a combination of the senolytic drugs dasatinib and quercetin (D+Q) reduces overall hepatic steatosis. Conversely, inducing hepatocyte senescence promotes fat accumulation in vitro and in vivo. Mechanistically, we show that mitochondria in senescent cells lose the ability to metabolize fatty acids efficiently. Our study demonstrates that cellular senescence drives hepatic steatosis and elimination of senescent cells may be a novel therapeutic strategy to reduce steatosis.

A tale of two paralogs: human Transformer2 proteins with differential RNA-binding affinities.

The Transformer2 (Tra2) proteins in humans are homologues of the Drosophila Tra2 protein. One of the two RNA-binding paralogs, Tra2ß, has been very well-studied over the past decade, but not much is known about Tra2α. It was very recently shown that the two proteins demonstrate the phenomenon of paralog compensation. Here, we provide a structural basis for this genetic backup circuit, using molecular modelling and dynamics studies. We show that the two proteins display similar binding specificities, but differential affinities to a short GAA-rich RNA stretch. Starting from the 6-nucleotide RNA in the solution structure, close to 4000 virtual mutations were modelled on RNA and the domain-RNA interactions were studied after energy minimisation to convergence. Separately, another known 13-nucleotide stretch was docked and the domain-RNA interactions were observed through a 100-ns dynamics trajectory. We have also demonstrated the 'compensatory' mechanism at the level of domains in one of the domain repeat-containing RNA-binding proteins.

Molecular mechanisms of mTOR regulation by stress.

Tumors are prime examples of cell growth in unfavorable environments that elicit cellular stress. The high metabolic demand and insufficient vascularization of tumors cause a deficiency of oxygen and nutrients. Oncogenic mutations map to signaling events via mammalian target of rapamycin (mTOR), metabolic pathways, and mitochondrial function. These alterations have been linked with cellular stresses, in particular endoplasmic reticulum (ER) stress, hypoxia, and oxidative stress. Yet tumors survive these challenges and acquire highly energy-demanding traits, such as overgrowth and invasiveness. In this review we focus on stresses that occur in cancer cells and discuss them in the context of mTOR signaling. Of note, many tumor traits require mTOR complex 1 (mTORC1) activity, but mTORC1 hyperactivation eventually sensitizes cells to apoptosis. Thus, mTORC1 activity needs to be balanced in cancer cells. We provide an overview of the mechanisms contributing to mTOR regulation by stress and suggest a model wherein stress granules function as guardians of mTORC1 signaling, allowing cancer cells to escape stress-induced cell death.

TSC1 activates TGF-ß-Smad2/3 signaling in growth arrest and epithelial-to-mesenchymal transition.

The tuberous sclerosis proteins TSC1 and TSC2 are key integrators of growth factor signaling. They suppress cell growth and proliferation by acting in a heteromeric complex to inhibit the mammalian target of rapamycin complex 1 (mTORC1). In this study, we identify TSC1 as a component of the transforming growth factor ß (TGF-ß)-Smad2/3 pathway. Here, TSC1 functions independently of TSC2. TSC1 interacts with the TGF-ß receptor complex and Smad2/3 and is required for their association with one another. TSC1 regulates TGF-ß-induced Smad2/3 phosphorylation and target gene expression and controls TGF-ß-induced growth arrest and epithelial-to-mesenchymal transition (EMT). Hyperactive Akt specifically activates TSC1-dependent cytostatic Smad signaling to induce growth arrest. Thus, TSC1 couples Akt activity to TGF-ß-Smad2/3 signaling. This has implications for cancer treatments targeting phosphoinositide 3-kinases and Akt because they may impair tumor-suppressive cytostatic TGF-ß signaling by inhibiting Akt- and TSC1-dependent Smad activation.

Applying genetic programming to the prediction of alternative mRNA splice variants.

Genetic programming (GP) can be used to classify a given gene sequence as either constitutively or alternatively spliced. We describe the principles of GP and apply it to a well-defined data set of alternatively spliced genes. A feature matrix of sequence properties, such as nucleotide composition or exon length, was passed to the GP system "Discipulus." To test its performance we concentrated on cassette exons (SCE) and retained introns (SIR). We analyzed 27,519 constitutively spliced and 9641 cassette exons including their neighboring introns; in addition we analyzed 33,316 constitutively spliced introns compared to 2712 retained introns. We find that the classifier yields highly accurate predictions on the SIR data with a sensitivity of 92.1% and a specificity of 79.2%. Prediction accuracies on the SCE data are lower, 47.3% (sensitivity) and 70.9% (specificity), indicating that alternative splicing of introns can be better captured by sequence properties than that of exons.

The testis-specific human protein RBMY recognizes RNA through a novel mode of interaction.

The RBMY (RNA-binding motif gene on Y chromosome) protein encoded by the human Y chromosome is important for normal sperm development. Although its precise molecular RNA targets are unknown at present, it is suggested that human RBMY (hRBMY) participates in splicing in the testis. Using systematic evolution of ligands by exponential enrichment, we found that RNA stem-loops capped by a C(A)/(U)CAA pentaloop are high-affinity binding targets for hRBMY. Subsequent nuclear magnetic resonance structural determination of the hRBMY RNA recognition motif (RRM) in complex with a high-affinity target showed two distinct modes of RNA recognition. First, the RRM beta-sheet surface binds to the RNA loop in a sequence-specific fashion. Second, the beta2-beta3 loop of the hRBMY inserts into the major groove of the RNA stem. The first binding mode might be conserved in the paralogous protein heterogeneous nuclear RNP G, whereas the second mode of binding is found only in hRBMY. This structural difference could be at the origin of the function of RBMY in spermatogenesis.

A class of human exons with predicted distant branch points revealed by analysis of AG dinucleotide exclusion zones.

BACKGROUND: The three consensus elements at the 3' end of human introns--the branch point sequence, the polypyrimidine tract, and the 3' splice site AG dinucleotide--are usually closely spaced within the final 40 nucleotides of the intron. However, the branch point sequence and polypyrimidine tract of a few known alternatively spliced exons lie up to 400 nucleotides upstream of the 3' splice site. The extended regions between the distant branch points (dBPs) and their 3' splice site are marked by the absence of other AG dinucleotides. In many cases alternative splicing regulatory elements are located within this region. RESULTS: We have applied a simple algorithm, based on AG dinucleotide exclusion zones (AGEZ), to a large data set of verified human exons. We found a substantial number of exons with large AGEZs, which represent candidate dBP exons. We verified the importance of the predicted dBPs for splicing of some of these exons. This group of exons exhibits a higher than average prevalence of observed alternative splicing, and many of the exons are in genes with some human disease association. CONCLUSION: The group of identified probable dBP exons are interesting first because they are likely to be alternatively spliced. Second, they are expected to be vulnerable to mutations within the entire extended AGEZ. Disruption of splicing of such exons, for example by mutations that lead to insertion of a new AG dinucleotide between the dBP and 3' splice site, could be readily understood even though the causative mutation might be remote from the conventional locations of splice site sequences.